The ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species are life- threatening superbugs due to their ability to escape the killing of traditional antibiotics. According to the Centers for Diseas Control and Prevention, these six types of bad bugs cause two thirds of the health care-associated infections, leading to 99,000 deaths annually in the United States. It is stunning that the annual frequency of deaths from Methicillin-resistant Staphylococcus aureus (MRSA) is comparable to those caused by HIV/AIDS. Therefore, there is an urgent need to develop new treatments against superbugs. Naturally occurring antimicrobial peptides are universal host defense molecules that have retained their potency throughout the years. To effectively exploit these interesting compounds, we have been constructing, expanding, and updating the widely used Antimicrobial Peptide Database (APD; http://aps.unmc.edu/AP). This comprehensive database facilitates naming, classification, statistical analysis, search, prediction and design of novel antimicrobials with desired properties. The APD tool has facilitated the research and education in the antimicrobial peptide field and laid a solid basis for this project. Based on our preliminary results, we hypothesize that most critical parameters can be extracted from the APD as a basis for designing and optimizing potent antimicrobial peptides that cause damage on bacterial membranes, leading to bacterial death and augmenting host defense. To test our hypothesis, we have designed the following specific aims: (1) To identify the critical parameters that determine potency of antimicrobial peptides and their mimics based on the APD; (2) To elucidate the critical modulator in antimicrobial peptides that determines mechanism of action and potential bacterial response genes; and (3) To examine the efficacy of database-designed peptides and their mimics against bacterial biofilm infection in vivo and mechanisms of immune modulation. To accomplish these aims, the PI has assembled a strong team that provides complementary expertise needed to understand host-pathogen interactions at the genetic, protein, and structural level as well as peptide-mediated immune responses in vivo using animal models. Because our database-designed compounds represent a novel antimicrobial strategy that effectively attenuated resistant superbugs both in vitro and in vivo, the outcome of this innovative research has great potential in providing potent antimicrobial agents that benefit patients.